Segmental pairs of dermal secretory cells release proteins into the hemolymph at the larval-pupal molt.

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.

Hepatopancreas immune response during molt cycle in the mud crab, Scylla paramamosain.

Molt is a critical developmental process in crustaceans. Recent studies have shown that the hepatopancreas is an important source of innate immune molecules, yet hepatopancreatic patterns of gene expression during the molt cycle which may underlie changes in immune mechanism are unknown. In this study, we performed Illumina sequencing for the hepatopancreas of the mud crab, Scylla paramamosain during molt cycle (pre-molt stage, post-molt stage, and inter-molt stage). A total of 44.55 Gb high-quality reads were obtained from the normalized cDNA of hepatopancreas. A total of 70,591 transcripts were assembled; 55,167 unigenes were identified. Transcriptomic comparison revealed 948 differentially expressed genes (DEGs) in the hepatopancreas from the three molt stages. We found that genes associated with immune response patterns changed in expression during the molt cycle. Antimicrobial peptide genes, inflammatory response genes, Toll signaling pathway factors, the phenoloxidase system, antioxidant enzymes, metal-binding proteins and other immune related genes are significantly up-regulated at the post-molt stage and inter-molt stage compared with the pre-molt stage, respectively. These genes are either not expressed or are expressed at low levels at the pre-molt stage. To our knowledge, this is the first systematic transcriptome analysis of genes capable of mobilizing a hepatopancreas immune response during the molt cycle in crustaceans, and this study will contribute to a better understanding of the hepatopancreas immune system and mud crab prophylactic immune mechanisms at the post-molt stage.

Dual role of the serine protease homolog BmSPH-1 in the development and immunity of the silkworm Bombyx mori.

Serine proteases and serine protease homologs are involved in the prophenoloxidase (proPO)-activating system leading to melanization. The Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization. Here, we show the dual role of BmSPH-1 in the development and immunity of B. mori. BmSPH-1 was expressed in hemocytes after molting and during the larval-pupal transformation in normal development. In contrast, following infection, BmSPH-1 was expressed in hemocytes and cleaved in the hemolymph, which resulted in the induction of PO activity. Moreover, BmSPH-1 was cleaved in the cuticle during the larval-pupal transformation and early pupal stages. In BmSPH-1 RNAi-treated silkworms, the reduced BmSPH-1 mRNA levels during the spinning stage or the prepupal stage resulted in the arrest of pupation or pupal cuticular melanization, respectively. The binding assays revealed that BmSPH-1 interacts with B. mori immulectin, proPO, and proPO-activating enzyme. Our findings demonstrate that BmSPH-1 paticipates larval-pupal transformation, pupal cuticular melanization and innate immunity of silkworms, illustrating the dual role of BmSPH-1 in development and immunity.

The transcription of atypical protein kinase C in hemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, during the molt stage and injection of pathogen-associated compounds.

Protein kinase C (PKC), which is involved in cell signaling pathways, comprises a family of serine/threonine kinases ubiquitously present in animals and its members are grouped on the basis of structural and activation characteristics into novel, classical, and atypical PKC forms. In this study, an atypical PKC of Macrobrachium rosenbergii, designated MraPKC, was successfully cloned, and its protein comprised structural domains similar to those of atypical PKC homologues, including the Phox and Bem1 (PB1) domain, a zinc finger phorbol-ester/DAG-type signature, protein kinase signatures, and a cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase C-terminal domain. Phylogenetic analyses revealed a close evolutionary relationship between MraPKC and aPKCs of insects. MraPKC transcripts were detected in all tissues examined through an RT-PCR, with the highest level detected in muscles. A quantitative real-time PCR was used to evaluate MraPKC expression in hemocytes of M. rosenbergii in various molt stages, and in prawn challenged with Vibrio alginolyticus, Lactococcus garvieae, and white spot syndrome virus (WSSV) as well as in prawns injected with pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PG), and polyinosinic:polycytidylic acid (Poly:IC). Results revealed that the expression pattern of MraPKC was distinctly modulated during molting, with significant enhancement in the C stage. MraPKC transcripts significantly increased in hemocytes of prawns infected with L. garvieae at 6-24 h and those injected with PG at 12-24 h. In contrast, significantly decreased expression of MraPKC was observed in hemocytes of prawns injected with V. alginolyticus and LPS for 3 and 12 h, respectively, and a similar phenomenon was observed in hemocytes of those injected with WSSV and Poly:IC for 12 h each. Therefore, MraPKC might play crucial roles in biological processes, and it may mediate the signaling pathway induced by varied pathogens for the potential regulation of host innate defense.

Molting-associated suppression of symbiont population and up-regulation of antimicrobial activity in the midgut symbiotic organ of the Riptortus-Burkholderia symbiosis.

The majority of insects possess symbiotic bacteria. Since symbiont titers can affect host phenotypes of biological importance, host insects are expected to evolve some mechanisms for regulating symbiont population. Here we report that, in the Riptortus-Burkholderia gut symbiosis, titers of the beneficial symbiont transiently decrease at the pre-molt stages in host development. This molting-associated suppression of the symbiont population is coincident with the increase of antimicrobial activity in the symbiotic midgut, which is observed in both symbiotic and aposymbiotic insects. Two genes, pyrrhocoricin-like antimicrobial peptide and c-type lysozyme, exhibit significantly increased expression in the symbiotic midgut at the pre-molt stages. These results suggest that the molting-associated up-regulation of antimicrobial activity in the symbiotic midgut represents a physiological mechanism of the host insect to regulate symbiosis, which is presumably for defending molting insects against injury and infection and/or for allocating symbiont-derived energy and resources to host molting.

An MBL-like protein may interfere with the activation of the proPO-system, an important innate immune reaction in invertebrates.

An important characteristic of the innate immune systems of crayfish and other arthropods is the activation of a serine proteinase cascade in the hemolymph, which results in the activation of prophenoloxidase and subsequently leading to the formation of toxic quinones and melanin. Although no true complement homologues have been detected in crayfish or crustaceans, several proteins with similarities to vertebrate pattern recognition receptors (PRRs), which are involved in the lectin pathway of complement activation in vertebrates, are present. One is a C-type lectin, a mannose-binding lectin (Pl-MBL), which is secreted from granular hemocytes. Here we report that Pl-MBL has LPS-binding capacity and is dependent upon high Ca(2+) for its solubility and Pl-MBL interferes with proPO activation in vitro when HLS is prepared at high Ca(2+). The proPO-activating system is efficiently activated by microbial polysaccharides and it has to be neatly regulated to avoid activation in places where it is inappropriate and the active enzyme PO should be prevented from spreading throughout the body of the animal. This may be particularly important during molting when proPO is involved in hardening of a new cuticle and the animal is vulnerable to microbes. The presence of high amount of Pl-MBL in the granular hemocytes may play a role in this process. Since a hemocyte lysate supernatant (HLS) prepared at 100 mM Ca(2+) could become activated when the concentration of LPS was increased up to 3 mg/ml, this may indicate that Pl-MBL acts as a scavenger for LPS to prevent spreading of LPS in the hemolymph to avoid further activation of the proPO-system.

[Specific immunity and polymorphism of breeding plumage in pied flycatcher (Ficedula hypoleuca) males (Aves: passeriformes)].

The relationship between the type of melanin-based plumage coloration and the strength of experimentally induced immune response was studied using as an example a pied flycatcher population from the Moscow Region. The plumage of pied flycatcher males exhibits the full spectrum of transitions from contrasting black-and-white to cryptic brownish, the latter being very similar to the coloration of females. In spite of numerous studies, the nature of this polymorphism still remains vague. Unlike many other avian species with monocyclic breeding, a considerable fraction of pied flycatchers combines two energy-consuming productive processes, breeding and molt. During the main experimental treatment we activated the humoral immunity of free-living males in chick-rearing period by injection of nonpathogenic multigenic antigen (sheep red blood cells, SRBC) and estimated the strength of the immune responses after repeated captures in 6-8 days. In addition, after each capture we estimated the numbers of leucocytes (WBC), heterophil to lymphocyte ratios (H/L) and measured night time basal metabolic rates (BMR). Non-molting males of different color types showed the same immune responses. Among molting birds, the strength of the immune response was significantly higher in pale males (morphs 4-7 by Drost's scale) than in bright males with rich melanin-based coloration (morphs 2-3). This difference resulted from two opposite processes. During molting, pale males heightened the antibody titer after immunization, while bright males tended to reduce the strength of immune response. Possibly such an asymmetry in immunocompetence at the first stage of molt reflects the different life strategies of pied flycatcher males - conspicuous birds less commonly combine breeding with molt than cryptic ones.

Effects of lipopolysaccharide on the expression of proinflammatory cytokines and chemokines and the subsequent recruitment of immunocompetent cells in the oviduct of laying and molting hens.

The goal of this study was to examine whether lipopolysaccharide (LPS) induces the expression of proinflammatory cytokines and chemokines and recruits T cells in the lower part of the oviduct, and whether that response to LPS is different between the laying and molting phase. White Leghorn laying and molting hens were intravenously injected with saline (control) or LPS. The uterus and vagina of oviducts were collected 3 or 6 h after injection, and used for reverse transcription PCR analysis of IL-1ß, IL-6, IL-8 (CXCLi2), and lymphotactin (Lptn), and for immunohistochemical analysis for the frequency of CD4+ and CD8+ T cells. The expressions of IL-1ß, IL-6, and CXCLi2 in the uterus and that of IL-1ß in the vagina were upregulated in response to LPS 3 or 6 h after injection in both laying and molting hens. The CXCLi2 expression in the vagina was upregulated by LPS in laying hens, whereas those effects of LPS were not significant in molting hens. Expression of Lptn showed a tendency to be downregulated after 3 h, with recovery by 6 h after LPS injection. The frequency of CD4+ T cells tended to increase in response to LPS after 6 h in the lamina propria of the uterus and vagina in both laying and molting hens. The CD8+ T cell frequencies in the lamina propria of the uterus and vagina of laying hens increased in response to LPS after 6 h. However, in the molting hens, LPS stimulation resulted in CD8+ T cell increase in the vagina only and not in the uterus. These results suggest that expressions of proinflammatory cytokines and CXCLi2 chemokine are upregulated in association with T cell recruitment in response to LPS in the lower part of the oviduct, although CD8+ T cells in the uterus may be depressed during the molting phase. These immunoresponses may play roles in the defense against infection of the oviduct.

Effects of immune activation and glucocorticoid administration on feather growth in greenfinches.

Elevation of glucocorticoid (GC) hormone levels is an integral part of stress response (as well as its termination) and immunomodulation. These hormones are also responsible for mobilizing energy stores by stimulation of gluconeogenesis and inhibition of protein synthesis. Elevation of GCs is thus incompatible with other protein-demanding processes, such as moult. Previous studies have shown that chronic elevation of GC hormones suppresses feather growth. Here, we asked whether similar effect would also occur in the case of acute GC elevation and induction of an inflammatory response by foreign antigen. We performed an experiment on captive wild-caught greenfinches (Carduelis chloris) injecting birds with phytohaemagglutinin (PHA) and dexamethasone (DEX) in a factorial design. To assess the possible somatic impacts of these manipulations, we removed one of the outermost tail feathers before the experiment and measured mass and rachis diameter and length of the replacement feathers grown in captivity. Immunostimulation by PHA reduced rachis length, but did not affect feather mass or rachis diameter. Single injection of a synthetic GC hormone DEX significantly reduced all three parameters of feather size. Altogether, these findings demonstrate the sensitivity of feather growth to manipulation of immune and adrenal functions. Our results corroborate the somatic costs of immune activation and suggest that even a short-term elevation of GC hormones may induce long-term somatic costs with a potential impact on fitness. Our findings also imply that a single injection of DEX, frequently used as a diagnostic tool, can have lasting effects and researchers must consider this when designing experiments.

Experimental test of a trade-off between moult and immune response in house sparrows Passer domesticus.

A trade-off between immune system and moulting is predicted in birds, given that both functions compete for resources. However, it is unclear whether such a trade-off exists during post-breeding moult. This study tests such a trade-off in the house sparrow (Passer domesticus). Males injected with an antigen (lipopolysaccharide) significantly moulted slower than sham-injected males. Moreover, males whose seventh primaries were plucked to simulate moult showed smaller immune response to phytohaemagglutinin than control males, in which seventh primaries were clipped. A trade-off between moult speed and body mass was also found. The results show a clear trade-off between moult and immune response in the house sparrow: immune response negatively affected moult and moult negatively affected immune response. These findings suggest that only individuals in good condition may have an efficient moult and simultaneously respond effectively in terms of immunity to pathogens, which could explain how plumage traits honestly indicate parasite resistance in birds.

Condition-dependent ecdysis and immunocompetence in the amphipod crustacean, Gammarus pulex.

The exoskeleton of arthropods forms an efficient protection against pathogens, but this first line of defence is periodically weakened during ecdysis, increasing the opportunity for surrounding pathogens to invade the body cavity. Since the richness of pathogens in the environment can be spatially and temporally variable, arthropods may have a fitness advantage in moulting in a place and time of low infection risk. Consistent with this hypothesis, we found that the amphipod crustacean, Gammarus pulex, exhibits temporal adjustment of the moult cycle in response to elevated risks of infection. Interestingly, this phenomenon is variable between two populations and independent of levels of immune defences. These results suggest that plasticity of the moult cycle in response to elevated risks of infection is adaptive and may result from adaptation to local variations in the risk of infection.

Immune-system activation depletes retinal carotenoids in house finches (Carpodacus mexicanus).

The costs of developing, maintaining, and activating the immune system have been cited as an important force shaping life-history evolution in animals. Immunological defenses require energy, nutrients and time that might otherwise be devoted to other life-history traits like sexual displays or reproduction. Carotenoid pigments in animals provide a unique opportunity to track the costs of immune activation, because they are diet-derived, modulate the immune system, and are used to develop colorful signals of quality. Carotenoids also accumulate in the retinas of birds, where they tune spectral sensitivity and provide photoprotection. If carotenoid accumulation in the retina follows the patterns of other tissues, then immune activation may deplete retinal carotenoid levels and impact visual health and function. To test this hypothesis, we challenged molting wild-caught captive house finches (Carpodacus mexicanus) with weekly injections of lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) over the course of 8 weeks. Immunostimulated adult males and females produced significant antibody responses and molted more slowly than uninjected control birds. After 8 weeks, immune-challenged birds had significantly lower levels of specific retinal carotenoid types (galloxanthin and zeaxanthin), but there were no significant differences in the plasma, liver or feather carotenoid levels between the treatment groups. These results indicate that immune-system activation can specifically deplete retinal carotenoids, which may compromise visual health and performance and represent an additional somatic and behavioral cost of immunity.

Utilizing fungus myceliated grain for molt induction and performance in commercial laying hens.

Molting in poultry is used to rejuvenate hens for a second or third laying cycle. Feed withdrawal was once the most effective method used for molt induction; however, it has being phased out due to food safety and animal welfare concerns. This study evaluated the utilization of fungus myceliated grain as a safe and effective alternative for inducing molt, enhancing immunity, reducing Salmonella growth, and returning to egg production. Laying hens were subjected to 1 of 5 treatments: 1) nonfed (NF), 2) full-fed (FF), 3) fungus myceliated meal (FM), 4) 90% fungus myceliated meal+10% standard layer ration (FM-90), and 5) 90% alfalfa meal+10% fungus myceliated meal (AF-90). Each treatment condition was replicated 9 times during a 9-d molt period. The results revealed that egg production for treatments 1 and 3 ceased completely by d 5, whereas hens in treatments 4 and 5 ceased egg production by d 6. The percentage of BW loss decreased significantly (P<0.05) in treatments 1 (57%), 2 (8%), 3 (35%), 4 (37%), and 5 (44%). Ovary weights of hens fed all molting diets decreased significantly from the full-fed control but did not differ significantly (P<0.05) from each other. Salmonella population in the crop, ovary, and ceca from hens differed significantly (P<0.05) among treatments. Return to egg production differed between treatments with higher production beginning in treatment 3 and ending in treatment 5. Antibody titers did differ (P<0.05) among treatments. From these results, fungus myceliated meal appears to be a viable alternative to conventional feed withdrawal and other methods for the successful induction of molt and retention of postmolt performance.

Molecular cloning and characterization of lipopolysaccharide- and beta-1,3-glucan-binding protein from the giant freshwater prawn Macrobrachium rosenbergii and its transcription in relation to foreign material injection and the molt stage.

Lipopolysaccharide (LPS)- and beta-1,3-glucan-binding protein (LGBP) complementary (c)DNA was cloned from the hepatopancreas of the giant freshwater prawn Macrobrachium rosenbergii using oligonucleotide primers and a reverse-transcription polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1389 bp encoding a protein of 378 amino acids (aa) including a 15-aa signal peptide. The calculated molecular mass of the mature protein (363 aa) was 41.2 kDa with an estimated pI of 4.73. The M. rosenbergii LGBP sequence contains (1) three putative integrin-binding motifs, (2) a glucanase motif, (3) a putative N-glycosylation site, (4) four protein kinase C phosphorylation sites, (5) four casein kinase II phosphorylation sites, and (6) a putative recognition motif. Sequence comparison showed that the deduced amino acids of LGBP of M. rosenbergii had overall similarities of 60-71% to those of known crustacean LGBPs and beta-1,3-glucan-binding proteins (BGBPs). The LGBP of M. rosenbergii was mainly expressed in the hepatopancreas. The LGBP transcript of M. rosenbergii was downregulated in haemocytes, but was upregulated in the hepatopancreas when injected with LPS and poly:IC after 12 h. The LGBP messenger (m)RNA expression of prawns in the postmolt stage was significantly upregulated in haemocytes, but downregulated in the hepatopancreas, which revealed a complementary relationship between haemocytes and the hepatopancreas in the molt cycle.

Is testosterone immunosuppressive in a condition-dependent manner? An experimental test in blue tits.

In this experiment we manipulated testosterone (T) and condition in juvenile male blue tits (Cyanistes caeruleus) during the moult, to test whether T's supposed immunosuppressive qualities are condition-dependent. To achieve this, we used T and control implants in combination with a dietary manipulation. We measured responses to both phytohaemagglutinin (PHA) and humoral immune challenges during the period of the treatments (moult) and also in the following breeding season (spring). During moult, males fed the enhanced diet were in better condition but there was no difference in humoral response between the dietary groups. T males produced a greater humoral antibody response than control (C) males. In the spring, males that had been previously treated with high T again exhibited higher antibody responses than C males. High T levels during moult were associated with a low PHA response but only in males with low body mass: heavier males that had high T exhibited the highest PHA responses. In the spring, the pattern of PHA responses was reversed; responses were highest in males that had low body mass but also had high T levels, and the lowest responses were by males that had both high T and were relatively heavy. Our results suggest that the effects of T on immunity can be either immunoenhancing or immunosuppressive, depending upon the condition of the individual, its life history stage, as well as on the immune challenge employed.

Identification and cloning of the alpha2-macroglobulin of giant freshwater prawn Macrobrachium rosenbergii and its expression in relation with the molt stage and bacteria injection.

The full-length complementary (c)DNA of the alpha2-macroglobulin (alpha2M) gene was cloned from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The 4875-bp cDNA contains an open reading frame (ORF) of 4419 bp, a 95-bp 5'-untranslated region (UTR), and a 361-bp 3' UTR containing the poly A tail. The ORF encodes a protein of 1472 amino acids (aa) with a 23-residue signal sequence. The molecular mass of the deduced amino acid sequence (1449 aa) was 163.29 kDa with an estimated pI of 4.88. The M. rosenbergii alpha2M sequence contains putative functional domains including a bait region and a GCGEQ internal thiol ester site, and a receptor-binding domain is present as in other aquatic arthropod alpha2Ms. Sequence comparison showed that alpha2M of this prawn had overall respective identities of 38.4%, 45.9%, 45.9%, and 46.0% to those of Scylla serrata, Litopenaeus vannamei, Penaeus monodon, and Marsupenaeus japonicus. A phylogenetic analysis revealed that M. rosenbergii alpha2M is the more-primitive genotype, and it showed significant differentiation from marine crustacean alpha2Ms. alpha2M was mainly expressed in haemocytes. The quantitative real-time RT-PCR analysis showed that alpha2M mRNA transcripts significantly increased in the A stage, achieved the highest level in the C stage, then declined in the D(0/1) stage, and reached the lowest level in the D(2/3) stage in haemocytes of prawn. alpha2M's expression in haemocytes of M. rosenbergii significantly increased at 24 h and 12 h after injection with heat-killed Lactococcus garvieae and Vibrio alginolyticus, respectively, which indicates that alpha2M is involved in the immune response of prawn.

A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.

High doses of dietary zinc induce cytokines, chemokines, and apoptosis in reproductive tissues during regression.

In chickens, high levels of dietary zinc cause molting, and the reproductive system undergoes complete remodeling concomitant to feather replacement. In the present study, the expression profiles of cytokines and chemokines were investigated in the ovary and oviduct of control hens and of hens induced to molt by zinc feeding. The zinc-induced feed-intake suppression, the changes in corticosterone levels, the immune cell populations in the reproductive tract, and the apoptosis of reproductive tissues were analyzed. The expression of mRNAs for interleukin-6 (IL-6), interferon-gamma (IFN-gamma), the avian ortholog of mammalian IL-8 (chCXCLi2), and a chicken MIP-1beta-like chemokine (chCCLi2) in the ovary and of mRNAs for IL-1beta, IL-6, IFN-gamma, transforming growth factor-beta2, chCXCLi2, and chCCLi2 in the oviduct were upregulated significantly during zinc-induced molting. A simultaneous feed-intake reduction was observed with higher expression of cytokines and chemokines. The results of the present investigation also suggested that the upregulation of corticosterone was closely associated with the increased expression of cytokines and chemokines. An increase in apoptosis within reproductive tissue during tissue regression was also noted. We had previously observed the upregulation of these cytokines expression in an earlier study (molting by feed withdrawal). However, the pattern and the level of expression were different among these two methods. These findings indicate that cytokines might be a common mediator of tissue regression during molting induced by diverse methods, although the pattern of induction is different. Thus, a high dose of dietary zinc seems to induce reproductive regression via the upregulation of cytokines and chemokines, the suppression of feed intake, and the increase in serum corticosterone, resulting finally in the apoptosis of reproductive tissues.

Dynamics of innate immune response in Gallus domesticus using two methods of induced molting.

The aim of this study was to compare the ability of laying hen abdominal macrophages during the second production cycle by using two different methods of induced molting. Two groups of Single Comb White Leghorn hens were induced to molt at the end of their first production cycle using feed restriction and ZnO supplementation. Macrophages were isolated from the abdomen and in vitro cytotoxic ability, at which point macrophage bactericidal moiety nitric oxide (NO) was recorded. Serum IgM and IgG titers against sheep red blood cells (SRBC) were determined at various stages: before molting (BM), 5% production level (5P), peak production stage (PP) and at the end of production (EP) level after fast and Zn-induced molt. Macrophages adherence percentage remained unaffected (p< or =0.05) during all production cycles, whereas the macrophage engulfment percentage and engulfment/cell was significantly higher (p< or =0.05) at PP in both fast and Zn-induced molted groups, as compared to all other studied stages. Macrophage NO production was increased (p< or =0.05) at PP and after SRBC and lipopolysaccrides (LPS) stimulus, when molted with ZnO supplementation. Serum total antibody titer against SRBC increased serum IgG and IgM titers during the second production cycle by Zn-induced molt. However, molting stress greatly reduced IgG and IgM production at the 5P stage. Serum Zn concentration increased with the onset of production but decreased at the EP stage irrespective of their molting regimes. Our results validate the strengthened innate and acquired immune response during the second production cycle after Zn-induced molting instead of fasting.

Cytokines in reproductive remodeling of molting White Leghorn hens.

The role of cytokines in regression of the ovary and oviduct during induced molting in chickens was investigated by evaluating the expressions of IL-1beta, IL-6, IFN-gamma, IL-2, TGF-beta2, MIP-1beta and IL-8 in the regressing ovary and oviduct by semi-quantitative RT-PCR. In addition, serum hormonal profiles (estrogen, progesterone and corticosterone), along with the gross regression and histological changes of the ovary and oviduct, were investigated. The correlation between expression of cytokines and hormonal changes during the induced molting was also studied. The expression of IL-6, IL-8, MIP-1beta and IFN-gamma mRNAs in the ovary, and IL-1beta, IL-6, IL-8, MIP-1beta, IFN-gamma and TGF-beta2 mRNAs in the oviduct, were up-regulated significantly during induced molting, suggesting their role in tissue regression. However, histological findings suggested no significant increase in immune cells in the regressing oviduct and ovary. Significant up-regulation of TGF-beta2 in the regressing oviduct might have suppressed leukocyte recruitment thereby preventing the inflammatory response and tissue damage. The down-regulation of estrogen and progesterone and up-regulation of corticosterone is well correlated with increased expression of cytokines. It appears that cytokines released during the process of induced molting may have a role in decreasing ovarian steroids and increasing the corticosterone levels in chicken. From this study, it may be concluded that cytokines play a major role in regression of the ovary and oviduct during induced molting in chickens.